title: Effects of apatite particle size in two apatite/collagen composites on the osteogenic differentiation profile of osteoblastic cells.
abstract: The development of new osteoconductive bone substitute materials is expected in medicine. In this study, we attempted to produce new hydroxylapatite (HAP)/collagen (Col) composites using two HAP particles of different sizes and porcine type I collagen. The two HAP particles were either nano-sized (40 nm in average diameter; n-HAP) or had macro-pore sizes of 0.5‑1.0 mm in length with fully interconnected pores (m-HAP). The aim of this study was to investigate the effects of apatite particle size in two HAP/Col composites on the osteogenic differentiation profile in osteoblast-like cells (SaOS-2). We created a collagen control sponge (Col) and two HAP/Col composite sponges (n-HAP/Col and m-HAP/Col) using freeze-drying and dehydrothermal cross-linking techniques, and then punched out samples of 6 mm in diameter and 1 mm in height. The SaOS-2 cells were cultured on three test materials for 1, 2, 3 and 4 weeks. Total RNA was extracted from the cultured cells and the expression of osteogenic differentiation-related genes was evaluated by reverse transcription PCR (RT-PCR) using primer sets of alkaline phosphatase (ALP), type 1 collagen (COL1), bone sialoprotein (BSP) and osteocalcin precursor [bone gamma-carboxyglutamate (gla) protein (BGLAP)] genes, as well as the β-actin gene. The cells were also cultured on Col, n-HAP/Col and m-HAP/Col specimens for 1 and 4 weeks, and were then observed under a scanning electron microscope (SEM). The experimental results were as follows: RT-PCR indicated that osteogenic differentiation, particularly the gene expression of BSP, was most accelerated when the cells were cultured on n-HAP/Col specimens, followed by m-HAP/Col, whilst the weakest accelaeration was observed when the cells were cultured on Col specimens. As shown by the SEM images, the SaOS-2 cells were fibroblastic when cultured on Col specimens for up to 4 weeks; they were fibroblastic when cultured on n-HAP/Col specimens for 1 week, but appeared as spheroids, while actively phagocytizing n-HAP particles at 4 weeks; however, they appeared as deformed fibroblasts when cultured on m-HAP/Col specimens, detached from the particles. Despite limited experimental results, our study suggests that n-HAP/Col may be employed as a new osteoconductive bone substitute material.
authors.affiliation: Department of Prosthodontics and Oral Implantology, Iwate Medical University School of Dentistry, Morioka, Iwate 020-8505, Japan.
authors.forename: Wataru
authors.initials: W
authors.lastname: Hatakeyama
authors.forename: Masayuki
authors.initials: M
authors.lastname: Taira
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Chosa
authors.forename: Hidemichi
authors.initials: H
authors.lastname: Kihara
authors.forename: Akira
authors.initials: A
authors.lastname: Ishisaki
authors.forename: Hisatomo
authors.initials: H
authors.lastname: Kondo
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001031:Apatites / Q000737:chemistry / Q000494:pharmacology*; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects* / Q000235:genetics; D002460:Cell Line; D003094:Collagen / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D053758:Nanoparticles / Q000737:chemistry; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology / Q000648:ultrastructure; D010012:Osteogenesis / Q000187:drug effects* / Q000235:genetics; D010316:Particle Size; D016062:Porosity; D034421:Sus scrofa; D054457:Tissue Scaffolds / Q000737:chemistry*
chemical_list: D001031:Apatites; D015415:Biomarkers; D003094:Collagen; D000469:Alkaline Phosphatase